Schizosaccharomyces pombe Git7p, a member of the Saccharomyces cerevisiae Sgtlp family, is required for glucose and cyclic AMP signaling, cell wall integrity, and septation

Abstract

The Schizosaccharomyces pombe fbp1 gene, encoding fructose-1,6-bisphosphatase, is transcriptionally repressed by glucose. Mutations that confer constitutive fbp1 transcription identify git (glucose-insensitive transcription) genes that encode components of a cyclic AMP (cAMP) signaling pathway required for adenylate cyclase activation. Four of these genes encode the three subunits of a heterotrimeric G protein (gpa2, git5, and git11) and a G protein-coupled receptor (git3). Three additional genes, git1, git7, and git10, act in parallel to or downstream from the G protein genes. Here, we describe the cloning and characterization of the git7 gene. The Git7p protein is a member of the Saccharomyces cerevisiae Sgtlp protein family. In budding yeast, Sgtlp associates with Skplp and plays an essential role in kinetochore assembly, while in Arabidopsis, a pair of SGT1 proteins have been found to be involved in plant disease resistance through an interaction with RAR1. Like S. cerevisiae Sgtlp, Git7p is essential, but this requirement appears to be due to roles in septation and cell wall integrity, which are unrelated to cAMP signaling, as S. pombe cells lacking either adenylate cyclase or protein kinase A are viable. In addition, git7 mutants are sensitive to the microtubule-destabilizing drug benomyl, although they do not display a chromosome stability defect. Two alleles of git7 that are functional for cell growth and septation but defective for glucose-triggered cAMP signaling encode proteins that are altered in the highly conserved carboxy terminus. The S. cerevisiae and human SGT1 genes both suppress git7-93 but not git7-235 for glucose repression of fbp1 transcription and benomyl sensitivity. This allele-specific suppression indicates that the Git7p/Sgtlp proteins may act as multimers, such that Git7-93p but not Git7-235p can deliver the orthologous proteins to species-specific targets. Our studies suggest that members of the Git7p/Sgt1p protein family may play a conserved role in the regulation of adenylate cyclase activation in S. pombe, S. cerevisiae, and humans.

FIG. 1.

Restriction map of insert DNA and git7 suppression analysis for plasmid pHF1 and related plasmids. Restriction sites for PstI (P), EcoRV (RV), NheI (N), and SpeI (S) are shown. git7 activity is based on the ability of the plasmids to restore glucose repression of fbp1-lacZ expression when introduced into strain CHP556 (git7-93) and to restore growth at 37°C to strain CHP718 (git7-235). β-Galactosidase activity (in parentheses) in CHP556 transformants represents the average specific activity and standard error for three independent transformants grown under glucose repression conditions and assayed twice each. Under the same conditions, strain CHP578 (git7⁺) carrying plasmid pHF5 possessed 5 ± 1 U of activity.

FIG. 2.

Amino acid sequence alignment of S. pombe Git7p and related proteins. The Git7p protein (GenPept accession number CAA19060) was aligned with the S. cerevisiae Sgt1p protein (ScSgt1p) (GenPept accession number NP_014700), the Oryza sativa Sgt1p protein (OsSgt1p) (GenPept accession number BAB19060), the Arabidopsis thaliana Sgt1p protein (AtSgt1p) (GenPept accession number T05589), and the human Sgt1p protein (huSgt1p) (GenBank accession number AF132856) by using the Clustal W (version 1.8) sequence alignment program (47); the alignment was displayed by using BOXSHADE. Identical residues are shown as white letters shaded in black, while conserved residues are shown as white letters shaded in gray. The methionine encoded by the putative start codon used by the partially functional, truncated git7 open reading frame carried on plasmid pHF4 (Fig. 1) is indicated by an asterisk. Alterations to the Git7p protein conferred by the git7-27, git7-93, and git7-235 alleles are also indicated.

FIG. 3.

Immunoblot of Git7p-V5 proteins. Protein extracts were prepared from CHP93 (git7-93) cells carrying either plasmid pKS1 (including 444 potential codons of git7) or plasmid pKS2 (including 379 potential codons of git7) and grown in the absence of thiamine (to promote expression from the plasmid-borne nmt81 promoter). Additional extracts were made from strains FWP101 (untagged git7⁺) and KSP5 (git7-V5 integrated at the git7 locus). Extracts were subjected to a Western blot analysis to detect V5-tagged (43) proteins. Lane 1, 20 μg of extract from CHP93/pKS1; lane 2, 7 μg of extract from CHP93/pKS2; lane 3, 60 μg of extract from FWP101; lane 4, 60 μg of extract from KSP5. Note that the CHP93/pKS1 extract in lane 1 contains a 54-kDa protein (indicated by an arrow) which was not seen in the other lanes and which may represent the protein translated from the first available start codon in pKS1.

FIG. 4.

Growth defects associated with git7 mutations. Cells from strains FWP101 (git7⁺; wild type), CHP27 (git7-27), CHP93 (git7-93), and CHP449 (git7-235) were grown for 24 h on YEA plates at either 30 or 37°C before being photographed.

FIG. 5.

Various morphologies associated with the git7-235 allele. CHP449 (git7-235) cells were grown for 24 h at 37°C, stained with Hoechst 33342 and Calcofluor, and photographed. Septa are indicated by arrowheads. (A) Cells displaying the wild-type phenotype, being either uninucleate or binucleate with a septum. (B) Multinucleate (m) and cell division-arrested (cd) binucleate cells. (C) Cells containing septa but failing to complete cytokinesis.

FIG. 6.

Starvation-independent sexual development in git7-GFP and git2Δ (adenlyate cyclase) homothallic strains. Cells of homothallic strains CHP795 (wild type), CHP792 (git7-GFP), and CHP558 (git2Δ) were pregrown at 37°C (to inhibit conjugation) in PM medium (8% glucose) to a concentration of 10⁷ cells/ml, diluted to 10⁶ cells/ml in the presence or absence of 5 mM cAMP, and incubated overnight at 30°C without shaking before being photographed.

FIG. 7.

Benomyl sensitivity in git7 mutant strains. Serial dilutions (1:5) of strains FWP101 (git7⁺), CHP27 (git7-27), CHP93 (git7-93), CHP449 (git7-235), CHP758 (git7-GFP), and CHP93 carrying plasmid pKS1 (git7-93 + git7-V5) were spotted on YEA plates containing 75 μg of adenine/ml and either 0 or 5 μg of benomyl/ml. Growth was recorded after 3 days at 25°C.

FIG. 8.

Localization of Git7p-V5. Indirect immunofluorescence was used to detect Git7p-V5 localization in KSP5 cells (carrying an integrated git7-V5 fusion) and in FWP101 cells (untagged git7⁺). Cells were stained with Hoechst 33342 to detect DNA prior to microscopy. KSP5 cells display a punctate signal throughout the nucleus and the cytoplasm. DIC, differential interference contrast microscopy.